Fluorescence imaging endoscope

ABSTRACT

The present invention relates to a fluorescence endoscope imaging system. The system uses first and second light sources to provide fluorescence and reflectance images of tissue being examined. An imaging device mounted at the distal end of the device is used to collect both images.

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation application of co-pending U.S.patent application Ser. No. 09/238, 664, filed Jan. 26, 1999, whichclaims the benefit of U.S. Provisional Application No. 60/072,455 filedon Jan. 26, 1998, the entire contents of which is incorporated herein byreference.

GOVERNMENT SUPPORT

[0002] The invention was supported, in whole or in part, by grantnumbers CA53717, P41RR02954, and DK 39512 from National Institutes forHealth. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The following relates to the development of a laser-inducedfluorescence imaging endoscope for mapping cancerous or precanceroustissues in hollow organs. In initial clinical studies, on colon polyps,Ultraviolet (UV) light was used at 370 nm to excite visible fluorescence(400-700 nm), the spectral signatures of which enabled differentiatingbetween normal and abnormal tissues. Previously endoscopic imaging hasbeen achieved using an optics module mounted in one of the biopsy portsof a two-port standard (white light) colonoscope. The optics moduleemploys a quartz optical fiber and associated optics to deliver the UVlight to the tissue, and a coherent quartz fiber-optic bundle totransmit the resulting fluorescence image to the proximal side of theendoscope, where a filter removes the large background of reflected UVlight and the fluorescence image is then captured by a high-gain CIDdetector array.

[0004] Endoscopically-collected autofluorescence images of colonicmucosa can be used as a screening tool for detecting pre-cursors tocolorectal cancer (CRC). Fluorescence has been used to distinguishbetween normal mucosa and adenomas. In particular, spectra measured withsingle point contact probes with the use of several different excitationwavelengths.

[0005] Fluorescence spectra have been obtained through optical fiberprobes with several excitation wavelengths. An in vitro study performeda search over a wide range of excitation wavelengths, and concluded that370 nm is optimal for distinguishing between normal mucosa and adenoma.Both in vitro and in vivo studies using adenomatous polyps as a modelfor dysplasia have shown that with this wavelength dysplasia has lesspeak intensity at 460 nm and may have increased fluorescence at 680 nmcompared with normal colonic mucosa. Furthermore, the morphologic basisfor these spectral differences has been studied by fluorescencemicroscopy. The decreased fluorescence intensity in polyps wasattributed to its raised architecture, increased vasculature, andreduced collagen in the lamina propria. The red enhancements arise fromincreased fluorescence of the crypt cells, which may be caused by higherlevels of porphyrin.

SUMMARY OF THE INVENTION

[0006] The present invention relates to imaging endoscopes and inparticular to a fluorescence imaging colonoscope using a dual channelelectronic endoscope that employs a charge coupled device (CCD) chip orother solid state imaging device mounted on its distal tip to collectthe white light image. Of particular significance for the presentinvention is that this chip can also collect the fluorescence image,displaying it on the endoscope's video monitor with much larger signalsize than that obtained using the optics module and intensified CIDcamera. This configuration was used to collect fluorescence images ofcolonic dysplasia. Video images of two small FAP polyps, have been takenwith the standard white light image and the unprocessed fluorescenceimage.

[0007] The CCD detector, which lacks gain intensification, detects theweak fluorescence signals, which are six orders of magnitude smaller inintensity than the diffusely reflected white light image. In addition,it is surprising that reflected 370 nm excitation light did notcompletely flood the CCD, obscuring the fluorescence signal. Thisresults from the fact that the CCD spectral response falls off to zeroquickly at wavelengths below 400 nm. Thus, the CCD effectively serves asits own long pass filter. Other imaging devices can be used with afilter to reduce by at least one half the detected intensity in theultraviolet region relative to the detected intensity in the visibleregion.

[0008] In this particular embodiment, the CCD has a resolution of270×328 pixels and an objective lens of 2.5 mm in diameter. The imagesare collected in 33 ms in RGB format. The advantages of this particularembodiment include that the in vitro fluorescence images exhibit asignal-to-noise ratio (SNR) of about 34 at clinical working distances of20 mm (distance between tip of endoscope and tissue surface), which issuperior to that obtained using the UV Module/CID detector, which has aSNR of about 18 at the same distance. The use of the CCD eliminates theneed for the optics module and greatly simplifies system design. Inaddition, it also avoids problems associated with the tendency of the UVmodule to rotate in the biopsy channel. By using the same detector andoptics for white light and fluorescence images, perfect registration ofthese two images can be obtained. Parallax between the white light imageof the CCD and the fluorescence image of the optics module was asignificant problem. The CCD in this particular embodiment contains88,560 pixels compared to 10,000 fibers for the UV module, resulting inhigher total image resolution. The objective lens on the Pentaxcolonoscope has better imaging properties than the UV module. Thecharacteristic width for the line spread function of the lens of thisembodiment is 200 mm compared to 400 mm for the UV Module. The overallrigidity of the spectral endoscope is not increased significantly with asingle UV illumination fiber.

[0009] The diagnostic methods employed can be based on the overallfluorescence intensity difference between normal mucosa and dysplasia.Thus, in certain applications it is preferable to collect thefluorescence emission over the full band between 400-700 nm. However,accurate measurements can use a point contact device such thatdiagnostic information can be obtained by sampling the fluorescence at aplurality of specific wavelengths such as 460, 600 and 680 nm, forexample. For many applications the preferred range for fluorescenceexcitation is between 350 nm and 420 nm. Endoscopic imaging studies withthe electronic CCD endoscope can include the use of color CCD's, whichhave the ability to provide such information.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a schematic view of an endoscopic system.

[0011]FIG. 2 is a schematic view of a solid state imaging device such asa CCD on the distal end of an endoscope.

[0012]FIG. 3 is a schematic diagram of an endoscopic system inaccordance with the invention.

[0013]FIG. 4 shows the relative sizes of the illumination area andfluorescence area.

[0014]FIG. 5 is a schematic diagram of an endoscopic system.

[0015]FIG. 6 is a graphical illustration of the average fluorescenceintensity and the measured and predicted signal to noise (SNR) ratio.

[0016]FIGS. 7A and 7B are graphical illustrations of variationfluorescence intensity between an average of 14 frames and a singleframe for normal colonic mucosa and adenoma, respectively.

[0017]FIG. 8 is an illustration of the sensitivity of the system as afunction of detection threshold values.

[0018]FIGS. 9 and 10 show fluorescence intensity profiles of tissue withadenoma, and including the moving average and percent ratio values.

[0019]FIG. 11 is the fluorescence intensity graph showing adenoma,normal and intensity ratio values as a function of pressure exerted onthe site with the probe.

[0020]FIG. 12 is an endoscope system showing the difference incollection geometry between the endoscope and a contact probe.

[0021]FIG. 13 is a preferred embodiment of an endoscope system inaccordance with the invention.

[0022]FIG. 14A is a preferred embodiment of a fluorescence imagingsystem in accordance with the invention.

[0023]FIG. 14B illustrates graphically the dependence of radiated poweron the input power of a light source emitting in the ultraviolet regionof the spectrum.

[0024]FIG. 14C illustrates a timing diagram for a process acquiringfluorescence and reference images.

[0025]FIG. 15 is a preferred embodiment of a fluorescence imaging systemin accordance with the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The equations describing the number of signal photons, N_(s),collected by a given pixel in an endoscope as a function of theseparation distance d and the radial distance p on the tissue surface,and the corresponding SNR are as follows: $\begin{matrix}\begin{matrix}{{N_{s}(p)} = \frac{\eta_{s}\lambda_{em}g^{2}T_{f}T_{i}T_{o}f_{p}r_{L}^{2}{ɛ_{t}( {\lambda_{em},{\Delta \quad \lambda}} )}\tan \quad \theta_{m}^{2}{P_{o}( \lambda_{ex} )}\Delta \quad t}{{hc}\quad 8( {1 - {\cos \quad \theta_{m}}} )N_{f}{d^{2}( {1 + ( \frac{p}{d} )^{2}} )}^{3.5}}} \\{{{SNR} = \frac{N_{s}}{\sqrt{N_{s} + ( \frac{\sigma_{e}}{G} )^{2}}}}\quad}\end{matrix} & (1)\end{matrix}$

[0027] The geometry and certain symbols are defined in FIG. 1. Note alsothe emission wavelength λ_(em), pixel array size g x g, fiber optictransmission efficiency T_(i), the bandwidth of the filtered emissionwavelength Δλ, the fraction of the transmitted energy in this wavelengthregion T_(f), the collective efficiency T_(o) of the system opticsincluding the long pass filter, lens and eyepiece, incident light energyP_(o)(λ_(ex))Δt, h is Planck's constant, c is the speed of light, f_(p)is the packing fraction of the fiber cores ε_(i) is the quantumefficiency of the tissue, and N_(f) is the total number of resolutionelements. The signal to noise ratio (SNR) is a function of electronicnoise σ_(e) and gain G.

[0028] Colorectal cancer constitutes a major national health careproblem. The incidence and mortality for carcinomas of the colon andrectum are second only to those of lung in the United States. Thissuggests that the current screening methods are inadequate forcontrolling the spread of colon cancer, and that little advancement indetection has occurred in a long time. The five year survival rate forall patients diagnosed is between 35-49%. Colorectal cancer isrelatively unresponsive to radiation and chemotherapy, hence surgicalresection with wide margins is the only reliable method of preventingits growth. These tumors spread by direct extension into adjacentstructures and by metastasis through the lymphatics and blood vessels.The most common sites of metastatic spread in order are regional lymphnodes, liver, lungs, and bones.

[0029] The pathophysiology of this disease begins in the epitheliallayer of colonic mucosa as dysplastic changes in the crypts cells. Thistissue can be accessed by colonoscope, and if the pre-malignant lesionsare detected at an early stage, they can be removed for biopsy. Mostcarcinomas of the colon and rectum are believed to arise from visibleprecursor lesions called adenomatous polyps. These benign masses evolvefrom a monoclonal expansion of epithelial cells which developirregularities in the size and shape of the nuclei and cytoplasm, acondition known as dysplasia. These lesions can be detected oncolonoscopy by their raised architecture. The medically acceptedadenoma-carcinoma sequence suggests that colorectal carcinoma arisesfrom adenomatous tissue that undergoes malignant transformation, whichis believed to occur through a multistep process in which geneticalterations accumulate. The presence of a precursor stage in thedevelopment of CRC provides a window of opportunity for early detectionand removal of these lesions to prevent future progression intocarcinoma.

[0030] The prevalent screening method of colonoscopy relies on theobservation of large structural changes in the colonic mucosa in orderto locate adenomatous tissue for biopsy. However, this procedure isrelatively insensitive to adenomatous tissue which is flat. Patientsdiagnosed with ulcerative colitis (UC), for example, have a high risk ofdeveloping carcinoma from non-polypoid regions of tissue. Moreoverrecent studies have concluded that some forms of adenocarcinoma arisefrom small superficial adenomas. Because of this risk, frequentscreening by colonoscopy must be performed with multiple biopsiesthroughout the colon. However, the likelihood of sampling error andmissed diagnoses in these patients renders this form of surveillancehighly unsatisfactory. Also, the examination of a tissue biopsy is timeconsuming and costly. Moreover, considerable intra- and inter-observervariation occurs in the identification of dysplasia. A patient who isdiagnosed as positive for dysplasia often must return to the clinic forfurther screening and possibly for surgical resection of the colon.Thus, the current state of endoscopic surveillance with histologicinterpretation is an imperfect science and is in need of improvedmethodologies with greater sensitivity and specificity and less intra-and inter-observer variation.

[0031] The method of fluorescence endoscopic imaging offers featureswhich can overcome the present screening limitations with white lightendoscopy. This method is sensitive to the biochemical constituents andmicroarchitecture below the tissue surface. Furthermore, combined withendoscopes, fluorescence images can scan wide areas, and can resolvetissue surfaces on the sub-millimeter scale. If sufficient informationis present on the fluorescence, computers can be used to determine thepresence and location of diseased regions in real-time. Autofluorescencehas demonstrated the ability to distinguish between normal andneoplastic human tissue. The first studies showed that single pointfluorescence spectra can be used to detect tumors in vitro from severaltypes of tissue. Later, in vivo studies were performed for detectingneoplasia in bladder, brain, colon, cervix, esophagus, lung, oralmucosa, and skin. In addition, fluorescence has been used to distinguishnormal tissue from diseased with the use of exogenous agent such ashematoporphyrin derivative (HpD).

[0032] The full length from the rectum to the cecum is typically 1.5 m.Histologically, the mucosa is the layer in contact with the lumen, andhas a thickness of about 400 μm. The epithelium is the most superficiallayer and consists of absorptive columnar cells and intermittentmucin-producing goblet cells, which function to reabsorb water and tolubricate. These cells undergo continuous turnover, and are replaced byrapidly dividing stem cells at the base of the crypts, where the firstsigns of dysplasia can be observed. The surrounding lamina propriacontains blood and lymphatic capillaries which supports the secretory,absorptive and other highly active functions of the mucosa. It consistsof loose connective tissue, in particular collagen, along with numerousinflammatory cells which protect the intestinal wall from invasion bymicrobes.

[0033] The muscularis mucosa is composed of several layers of smoothmuscle fibers which contract to expel secretions from the glandularcrypts, prevents clogging, and enhances absorption by maintainingcontact between epithelium and luminal contents. The submucosa containsthe larger blood vessels, lymphatics, and nerves, and are surrounded bydense collagenous connective tissue which keeps the mucosa attached tothe muscularis propria. The muscularis propria contains an innercircular and outer longitudinal muscle layer, which are involved in theinvoluntary peristaltic contractions of the colon for propagating theflow of fecal matter. The outer serosal layer consists of connectivetissue which contain the major blood vessels and nerves.

[0034] Adenomatous polyps are raised protrusions of mucosa which containimmature, poorly differentiated epithelial cells with irregularity insize and shape of the nuclei. These lesions are benign but they have thepotential to transform into colorectal carcinoma. The differentmorphological types include tubular, villous, and tubulovillousadenomas. Although all forms are raised, each type can either contain astalk, which is called pedunculated, or can be hemispheric, which isknown as sessile. The malignant potential of polyps are greatest withthe villous form and least with the tubular. Also, the probability ofcarcinoma developing increases with the size of the polyp. There isabout a 1% chance of finding invasive tumor in a polyp less than 1 cm indiameter, 10% for polyps between 1 and 2 cm, and 45% for polyps largerthan 2 cm. The sub-cellular changes associated with these polyps arefrequently histologically identical to the dysplasia found in ulcerativecolitis.

[0035] Results of molecular biology studies suggest that the stepsinvolved in the malignant transformation of adenoma into carcinomainvolves the mutational activation of an oncogene coupled with thesequential loss of several tumor suppressor genes. Also, it was foundthat several genes must incur mutations before malignant tumors anse.Several specific genetic alterations have been identified during theprocess of tumorigenesis. Activational mutations have been found in theras oncogene of 50% of colorectal carcinoma. Furthermore, allelicdeletions were identified in portions of chromosomes 5, 17, and 18,which may involve loss of tumor-suppressor genes.

[0036] Patients with the presence of over 100 neoplastic polyps in theircolon are diagnosed with the condition called familial adenomatouspolyposis. These people have a genetic predisposition for developingnumerous polyps in their colon by adulthood. Most patients have between500 and 2500 polyps, and on average, there are about 100 polyps. FAP isa rare disease, and accounts for only about 1% of the incidence of CRCin the Western world. Foci of dysplasia usually become malignant, andFAP patients must have their colons removed at a young age. Theprobability for the onset of colon cancer for someone with thiscondition is 10% at 10 years of age, 50% at 20 years, and 100% at age30. Histologically, most of the polyps are tubular adenomas with a highprobability of malignant transformation, and the dysplasia associatedwith FAP polyps is identical to that found in sporadic polyps. Anautosomal dominant genetic defect is responsible for the development ofthis disease.

[0037] A second form of CRC that is associated with familialpredisposition is hereditary nonpolyposis colorectal cancer (HNPCC).HNPCC is defined as patients with at least three relatives in twogenerations having CRC, and with at least one relative being diagnosedat less than 50 years old. This form is much more common than FAP, andaccounts for up to 13% of the incidences of CRC in the Western world.HNPCC patients do not have numerous adenomatous polyps, and it is verydifficult to distinguish it from sporadic cases. Genetic linkage hasbeen found between this disease and anonymous microsatellite markers onchromosome 2.

[0038] In ulcerative colitis, the mucosa undergoes cytological changesresulting in the formation of dysplasia without the presence of polypformation. These changes are believed to be associated with repeatedepisodes of chronic inflammation and repair of the colonic epithelium,and flat, ulcerated tumors with poorly defined margins are common.Patients who have had UC for over 8 years are recommended to haveperiodic colonoscopy with random biopsies taken. This screening processis not effective because less than 0.1 percent of the total mucosalsurface area is sampled. However, it is important to note that only 1%of new incidences of CRC arise from UC cases.

[0039] UC is an inflammatory disorder of the colorectal mucosa ofunknown cause. Patients with UC are at increased risk for developingdysplasia or cancer. Recognition of this increased risk has resulted incolonoscopic surveillance strategies starting at 7-10 years after theinitial presentation of symptoms. Colonic surveillance strategiesinclude direct macroscopic visualization of colonic mucosa and access tomucosal biopsies for microscopic assessment of dysplasia. Although thepathological classification of dysplasia was standardized in 1983,differences and inconsistencies remain regarding the interpretation ofdysplasia.

[0040] Dysplasia is typically focal. Despite the practice of taking12-20 mucosal biopsies during surveillance colonoscopy, less than 1% ofthe colonic surface is sampled, so the likelihood of missing small fociof dysplasia is high. Thus, cancers can develop in patients without anyprevious or concurrent dysplasia. Although performing prophylacticcolectomy on all patients after the first decade of disease would be themost definitive solution to the cancer problem in UC, patients withminimal or mild symptoms of the disease are understandably reluctant totake this radical approach. Colonoscopic surveillance with histologicinterpretation remains an imperfect science in need of improvedmethodologies with greater sensitivity and specificity.

[0041] Furthermore, studies have suggested that flat dysplasia may bethe origin of sporadic colon cancer which does not arise via theadenoma-carcinoma sequence. The morphological characteristics ofadenomas that proliferate superficially in flat nonpolypoid mucosa havebeen observed endoscopically as small plaquelike lesions with vagueredness or discoloration. In a comprehensive study, 33 such lesions weredescribed as slightly elevated with a reddish surface and a centraldepression. Foci of cancer or severe atypia were found in 25% of lesionsof diameter up to 4 mm, 40% of lesions measuring between 5 and 8 mm, and80% of lesions with diameter between 9 and 10 mm.

[0042] There are several methods in practice for the early screening forCRC, but each is limited in its effectiveness. The goal of screening isto detect localized superficial masses in asymptomatic individuals.

[0043] A sigmoidoscopy involves the clinician viewing the patient'srectum and sigmoid colon with either a rigid or flexible imaging device.This form of screening is based on the finding that 60% of CRC occurwithin the distal 25 cm of the colon. This length is reachable with arigid sigmoidscope, and a flexible one can reach up to 60 cm. However,recent statistics have shown that an increasing number of tumors arefound beyond the reach of this device. An advantage of this procedure isthat it can be performed without the patient undergoing anesthesia ortaking a prep. The most extensive method of screening for this diseaseis a colonoscopy, where the patient is first prepped and sedated. Acolonoscope is inserted throughout the full length of the colon, and themucosal surface is viewed by the physician under white light for polypsand other abnormal masses. This procedure is adequate for identifyingraised lesions, but flat region of dysplasia will go undetected.

[0044] The fluorescence of tissue occurs through a process in which theelectrons of a biological molecule enters an elevated energy state uponabsorbing laser light at a given excitation wavelength λ_(ex). Theexcited state is unstable, and the electrons will return to the groundstate. Most of this energy is lost as heat through molecular collisions,but a small fraction of excited electrons undergo an internal conversionand spontaneously radiates light at longer emission wavelengths λ_(em).The fraction of molecules which release energy by fluorescence is calledthe quantum efficiency of the tissue, denoted as ε_(t). The fluorescenceintensity depends on the product of the initial population of theexcited state and the tissue quantum efficiency.

[0045] The spectral lineshape is determined by the fluorescence emissionand absorption by biochemical molecules which are unique to thecomposition of tissue. The electronic levels of the single state aresplit into vibrational and rotational states, which in large moleculesconsists of small intervals and may overlap due to molecularinteractions. The electrons may decay to any of thevibrational-rotational levels of the ground state; thus, thefluorescence spectra of biomolecules are typically broad. This lack ofstructure in the spectra limits the amount of information that can beobtained from fluorescence. The tissue components which producefluorescence are known as fluorophores, and endogenous chromophoresinclude aromatic amino acids, NADH, FAD, and porphyrins. The localenvironment may have a large effect on the fluorescence emission, whichmay become quenched or shifted in wavelength. Further details regardingthe use of outafluorescence for imaging tissue can be found in U.S. Pat.Nos. 4,193,142, 5,421,337, 5,452,723, 5,280,788 and 5,345,941, theentire contents of these patents being incorporated herein by reference.

[0046] A first step taken in evaluating the use of fluorescence in colonwas to determine the existence of optimal wavelengths to differentiatebetween normal colon mucosa and adenomatous polyps in vitro with singlepoint measurements on a sub-millimeter scale. For example, thefluorescence emissions of 4 normal colon and 11 adenomatous polyps wererecorded with a spectrofluorimeter. The excitation wavelengths usedranged between 250 to 500 nm in 10 nm steps, and the results weretabulated in an array called an excitation-emission matrix (EEM). Aratio was taken of the average EEM from the normal colon to that of theadenomatous polyps, and excitation at 330, 370 and 430 nm were found toproduce fluorescence spectra which contained the greatest amount ofdiagnostic information.

[0047] Based on the results of these in vitro studies, clinical trialswere conducted to evaluate the ability of fluorescence to distinguishamong normal, adenomatous, and hyperplastic colon tissue with 370 nm. Inthis study, a pulsed nitrogen-pumped dye laser delivered 370 nmexcitation through an optical fiber probe with one excitation and sixcollection fibers. This probe was inserted through the biopsy channel ofa colonoscope, and placed in contact with the colonic mucosa duringcolonoscopy. The probe consisted of six individual 200 μm collectionfibers arranged in a bundle with one fiber for excitation. With thisdevice, fluorescence emission was detected from an area of tissue 2about 1 mm . The fluorescence spectra were detected by a spectrographcoupled to an OMA. The spectra showed a difference at 460 nm where thenormal mucosa produced about 6 times greater fluorescence intensity thanadenoma. This difference is almost twice that found from the in vitrostudies. Above 650 mn, the average of the adenomas were slightly greaterthan that of normal.

[0048] From 20 patients, the fluorescence intensities at 466 and 680 nmwere located on a scatter plot, and a straight line was drawn tominimize the number of misclassifications when compared to histology.The decision line correctly classified 31 of 31 adenomas, 3 of 4hyperplastic polyps, and 31 of 32 normal colonic tissue specimens. Thesensitivity, specificity and positive predictive value of the techniquefor diagnosing adenomas were 100%, 97%, and 94%, respectively. Becauseonly a small number of hyperplastic polyps were examined, it was unclearwhether adenoma could be reliably distinguished from hyperplasia usingfluorescence. The observed differences in the fluorescence may arisefrom architectural differences between polyps and the normal mucosarather than from dysplastic changes.

[0049] The next step was to use the data from this study to provideprospective methods of evaluating the performance of fluorescence. Thedata were randomly divided into two equal sets, and the first was usedto devise an algorithm to distinguish the tissue type based on thefluorescence intensity at 460 nm and at the ratio between intensities at680 to that at 600 nm. A biopsy of tissue from each point was classifiedhistologically as adenomatous, hyperplastic, or normal. From theprospective decision criteria, the sensitivity, specificity and positivepredictive value of the algorithm for diagnosing adenomas were 90%, 95%,and 90%, respectively.

[0050] Further attempts have been made to use fluorescence todistinguish between normal mucosa, adenomatous polyps and hyperplasticpolyps in vivo with 337 nm excitation. Fluorescence spectra weremeasured from 86 normal colonic sites, 35 hyperplastic polyps, and 49adenomatous polyps with a single optical fiber. The fluorescenceemission displayed peaks at 390 and 460 nm, which was attributed to thecollagen in the submucosa. Also, this peak decreased in intensity fornormal mucosa, hyperplastic polyps, and adenomas, respectively. The peakintensity of the normal mucosa was found to be slightly less than twicethat for adenomas. Using a MVLR analysis, the sensitivity, specificity,positive predictive value and negative predictive value of fluorescenceto distinguish between adenomatous and hyperplastic polyps were 86%,77%, 86% and 77%, respectively. This study concluded that thedifferences in fluorescence were due to polyp morphology rather than tothe fluorophores present in the polyps.

[0051] Other excitation wavelengths have been used to study fluorescencein colon. A continuous wave He—Cd laser was used to deliver 325 nmexcitation to measure fluorescence spectra from 35 normal mucosa and 35adenomatous polyps in vitro from a single optical fiber by an OMA. Thepeak intensity from normal mucosa occurred at 375 nm and that foradenoma appeared at 450 nm. A multi-variate linear regression (MVLR)analysis established a set of scores for each data point to determine adiagnostic criterion. Fluorescence spectra from an additional 34 normal,16 adenomatous, and 16 hyperplastic sites were taken and analyzedprospectively using the established decision criteria. The sensitivity,specificity and positive predictive value of this study to distinguishbetween normal and adenomatous tissue were found to be 100%, 100%, and94%, respectively. In addition, 15 of 16 hyperplastic polyps wereclassified as normal, which is the correct diagnosis becausehyperplastic polyps are formed from a thickening of the epitheliallayer.

[0052] The fluorescence of colon was studied with 410 nm excitation aswell. The emission from 450-800 nm was collected with aspectrofluorimeter from 83 biopsy specimens removed during colonscopyfrom 45 patients. The intensity of the emission band from 460-530 nmdeclined from normal to carcinoma to adenomatous mucosa. The peakintensity at 460 nm was about 2.5 times higher for normal mucosa thanfor adenoma. A stepwise discriminant analysis was performed on thespectra using nine variables. The results compared to histology showedthat the process distinguished between normal mucosa and adenoma with asensitivity and specificity of 88.2% and 95.2%, respectively. Thefluorescence emission resulted from the superposition of three bandscentered at about 470, 485, and 404 nm.

[0053] Thus, there has been extensive research performed within the last5 to 10 years to evaluate the use of tissue autofluorescence todistinguish between adenomatous and normal mucosa. In vitro studies haveconcluded that 330, 370, and 430 nm are optimal for excitation.Preliminary in vivo results indicate that single point fluorescencedetection has a sensitivity, specificity, and positive predictive valueas high as 90%, 95% and 90%, respectively for such discrimination. Also,the data suggest that the intrinsic fluorophores can include collagen,NADH, and porphyrin. Hemoglobin is an absorbing chromophore. In order tomake this technique suitable for a clinical setting, wide areafluorescence detection and processing must be performed in real time andadapted to conventional white light endoscopy. These requirements demandthe development of a spectral imaging instrument.

[0054] Fluorescence microphotographs of unstained frozen sections werestudied to account for the morphological structures in normal colonicmucosa and adenomatous polyps which emit fluorescence. The 351 and 364nm lines from an argon-ion laser were used for fluorescence excitation,and the emission was collected by a series of barrier filters withcut-off wavelengths of 420, 475, 515, 570 and 610 nm. The fluorescenceintensity was graded semi-quantitatively from 1+to 4+by a singleobserver. In normal mucosa, fluorescence in the spectral band from 420to 700 from collagen in the lamina propria was graded at 3+ and that inthe submucosa at 4+in the same emission bandwidth. In the epithelium,there was faint fluorescence seen from absorptive cells and none fromgoblet cells. The H&E stained section identifies the tissue compositionof normal mucosa. The fluorescence image of the serial unstained sectionindicated the fluorescent structures. Several differences were observedon the fluorescence image of the serial unstained section indicating thefluorescent structures.

[0055] Several differences were observed on the fluorescence micrographsof the adenomatous mucosa. First, fewer collagen fibers were present inthe lamina propria, resulting in less fluorescence intensity from theepithelium. Also, the level of fluorescence seen in the cytoplasm ofcrypt cells was recorded at 2+, compared to +/− seen in normal crypts.Finally, a larger number of fluorescent granules were present inadenoma. The image of the H&E stained section include crypt cells froman adenomatous polyp. The fluorescence from the serial unstained sectionshows an observable level of fluorescence, and the number of eosinophilsin the lamina propria is significantly larger than that in normalmucosa. The submucosa of the adenomatous polyp was graded at 4+, whichis the same as that of normal.

[0056] A procedure has been developed to describe the clinicallyobserved fluorescence in terms of its microscopic origins. This processcombined the intrinsic fluorescence of each microstructure with itsdensity as a function of tissue depth and the optical turbidity of theincident and return path. The concentrations of each fluorophore fromclinical fluorescence spectra can then be extracted. From thisprocedure, the factors for observing greater fluorescence intensity fromnormal mucosa compared to that from adenomas include: (1) The submucosalfluorescence is about 10 times brighter than that of the overlyingmucosa. (2) The mucosa attenuates both the incoming excitation light andthe returning fluorescence; if the mucosa is sufficiently thick, theunderlying submucosa cannot contribute, but if it is thin, as in normalmucosa, attenuation is smaller, resulting in brighter tissuefluorescence. (3) In addition, the fluorescence intensity of adenomas isless than that of normal colonic mucosa, perhaps because the dysplasticcrypts tend to displace the collagen in the lamina propria, which is thedominant fluorophore. (4) Adenomas exhibit greater attenuation of boththe 370 nm excitation light and the return fluorescence, due toincreased hemoglobin-rich microvasculature.

[0057] A multi-spectral imaging system has been developed which collectsfluorescence at four different emission wavelengths simultaneously. Inthis device, the output of a fiber optic endoscope is passed through 4spatially separated interference filters. The 4 images are arranged ontoquadrants of an intensified CCD array by adjustable segments of amulti-mirror system. The CCD or other imaging device 40 as seen in FIG.2 can have 30,000 pixel elements or more. The four wavelengths wereselected to optimize the contrast in the fluorescence spectra betweennormal and diseased tissue. Fluorescence from human cadaveric aorta wasexcited with 337 nm, and emissions from 400, 420, 450, and 480 nm wereratioed to produce a dimensionless contrast function. This functionindicated a value for atherosclerotic plaque that was four times greaterthan that for normal artery, and the results were displayed using afalse-color overlay. This instrument was also able to distinguishbetween rat tumor and surrounding muscle from fluorescence spectra.Further details regarding this system are described in Wang, T. D. et.al., “Real-time In vivo Endoscopic Imaging of Fluorescence from HumanColonic Adenomas”, proceeding of SPIE 1998, 3259, the entire contents ofwhich is incorporated herein by reference.

[0058] The resolution of this design is limited by the fibers in theimaging bundle. The use of 4 fluorescence emission wavelengths providesfor greater contrast between normal and diseased tissue and forflexibility in the development of the disease detection process.However, by separating the fluorescence emission in parallel, the signalis reduced by a factor of 4, thus lowering the SNR. Also, the 4 spectralimages must be aligned onto the detector at different angles, whichposes a challenge for image registration. Furthermore, image processingalgorithms using multiple images increase the computation time, and itis not clear that the fluorescence contains independent information at 4bands. Finally, the fluorescence images are detected at the proximal endof the endoscope, which poses difficulty in clinical use for registeringthe white light image and in navigating the instrument.

[0059] A simpler version of the multi-spectral imaging system has beendeveloped which collects only 2 emission bands. This design splits thefluorescence emission with a beam splitter onto two intensified CCDcameras. A helium cadmium laser delivers excitation light at 442 nm viathe illumination bundle of a fiberoptic bronchoscope. The fluorescenceemission was filtered in 2 bands, one between 480 and 520 nm and theother at wavelengths greater than 630 nm. The two spectral images werealigned, and the intensities were ratioed point by point fordiscriminating normal from diseased tissue, and a color image wasformed. This method eliminates the effects of distance and angle of theilluminating light, as well as tissue reflective properties. A colorcamera is attached separately for observing the white light image. Thissystem was tested clinically on 53 patients and 41 volunteers, and theresults were compared with conventional white light bronchoscopy at 328sites. The sensitivity on fluorescence was 73%, which was significantlygreater than that of 48% found on white light in detecting dysplasia andcarcinoma in situ. The two methods were found to have the samespecificity of 94%.

[0060] In the clinical system, the white light and fluorescence imageswere collected with a dual-channel electronic colonoscope (PentaxEC-3800TL). This model contains two biopsy channels with diameters of3.8 and 2.8 mm, respectively. The outer diameter of the endoscope is12.8 mm, and the working length in 1.7 m. The field of view of themulti-element objective lens has a divergence half-angle of 60° with adepth of focus ranging between 5 and 100 mm. The white lightillumination is produced by a 300 W short-arc xenon lamp. By using thesame detector for both white light and fluorescence imaging, perfectregistration can be obtained. This feature is ideal for producing adiagnostic overlay.

[0061] An illumination probe consisted of 200 μm core diameter opticalfiber with NA 0.40 coupled to a 3 mm diameter BK7 glass microlens(F#=-1). The illumination probe was inserted into one of the instrumentchannels, and the tip was placed flush with the distal end of thecolonoscope. A mode mixer clamped the excitation fiber at the proximalend to maximize the divergence angle of the light. The probe wasattached at the proximal end of the colonoscope by a leur lock toprevent movement. A power of 300 mW was delivered to the tissue. Thespectral response of the CCD detector (TI TC210) cuts off at about 400nm, and is negligible at the excitation wavelengths λ_(ex)=351 and 364nm³, thus eliminating the need for a long pass filter to block specularreflection from the excitation light. The two instrument channels allowfor the optical fiber illumination probe and the bioposy forceps to beused at the same time. FIG. 1 shows a schematic view of the endoscope 10with an imaging bundle 20, bioposy view of the endoscope 10 with animaging bundle 20, biopsy channel 12, lens 18, and illumination ports14. The distal end of the device is positioned at a distance d from thetissue. One problem associated with such a system is the shadowsgenerated by the illumination system. An important feature of theinvention described below is a process to compensate for shadows on thetissue 16 surface.

[0062] A footswitch was activated by the user to block the excitationlight when the white light was used for illumination, and vice versa,using a pair of computer controlled shutters (Uniblitz, VS 14). Theintegration time for acquiring each fluorescence image is 33 ms. Asshown in FIG. 3, the clinical fluorescence imaging system 40 consists ofa video processor 48, computer 44, monitor 46, mavigraph 50, and VCR 52,laser 42 and colonoscope 54.

[0063] An electronic colonoscope 54 detects photons at the distal endwith a CCD detector. An important aspect of the present invention isthat the spectral response of the Texas Instrument TC-210 CCD detectordropped sufficiently fast below 400 nm that no diffuse reflection fromthe UV excitation was observed. In fact, virtually no specularreflection, which is several orders of magnitude higher in intensitythan diffuse reflectance and fluorescence, was observed either. Anotheraspect which made this system possible was that the detector hassufficient sensitivity to detect fluorescence from colonic mucosawithout the use of an intensifier. Because the detector is located atthe distal end, the optical transmission efficiency is determined onlyby the multi-element objective lens positioned between the detector andthe tissue. Another significant feature of this embodiment of theinvention is that the same chip detects both the white light andfluorescence image, thus perfect registration occurs on the pseudo-coloroverlay. Furthermore, no modifications are necessary to the colonoscopewhich can impede the clinician's ability to perform the procedure.

[0064] One limitation of this system is the bandwidth selectivity andspectral resolution of the chip. The TC 210 is a monochrome detector andcollects fluorescence over the full visible spectrum. It is difficult toemploy bandpass filters in front of the CCD because the light iscollected at angles at high as 60°. However, RGB detectors exist whichcontain pixels which are sensitive to red, green, and blue light, andcan produce fluorescence images in 3 frames. However the passbands aredetermined by the integrated circuit manufacturer of the imagingcircuit. Note that a gating mechanism can also be used, which isdesirable for using pulsed lasers as the excitation source. Otherexcitation sources can include CW lasers and broad or narrow band lightsources.

[0065] The block diagram of an electronic imaging system operated byswitch 76 is shown in FIG. 5. An argon-ion laser 60 delivers UV lightthrough a shutter 62 into a quartz optical fiber coupled to a microlenslocated in one instrument channel of the colonoscope, while the whitelight 64 is delivered through shutter 66 the illumination fibers of port70. The pair of shutters 62, 55 are computer-controlled by a digitalinput/output (I/O) card 74. Both the fluorescence and white light imagesare detected by the CCD 72 at the distal end. A frame grabber 78digitizes the fluorescence and white light images sequentially. A hostmicrocomputer executes the image processing algorithm and displays thepseudo-color overlay. A mavigraph is used to convert the white lightimage with overlay into a format which can be recorded by the VCR.

[0066] The plot in FIG. 6 shows the fluorescence intensity from theaverage of 14 frames collected with the electronic imaging system. A rowof pixels is shown from normal colonic mucosa. Also plotted are themeasured and the predicted SNR. The SNR is approximately 30 at thecenter and it falls to about 10 near the periphery. Thus, the full fieldof view satisfies the minimum SNR requirement of 4 for theinstrument-noise limited detection for distinguishing between normalcolonic mucosa and adenomas.

[0067] A frame-to-frame variation from average in the fluorescence imageintensities can be seen in FIGS. 7A and 7B, which show the differencesbetween the values across a row of pixels in a single frame compared tothe average of 14 frames. The plot in FIG. 7A is that for the normalspecimen shown in FIG. 6, and the plot in FIG. 7B is from a sample ofmucosa which contains an adenoma in the center. The variation about theaverage is small compared to the difference in fluorescence intensitybetween normal and adenomatous tissue. Thus, the occurrence of falsepositives resulting from pixel-to-pixel variation is small.

[0068] A streaking artifact appeared in the fluorescence images takenwith the electronic imaging system. This artifact arose because the UVexcitation light was not blocked with the CCD rows were being read outelectronically, which is performed under normal white light illuminationby a rotating wheel with spatially separated filter. This artifact canbe removed in the processing software of the image data.

[0069] A study was performed to determine the level of UV light whichcan be safely delivered onto the colonic mucosa. White light andfluorescence images were collected sequentially. Fluorescence imagesfrom 30 patients with 14 colonic adenomas and 6 hyperplastic polyps werecollected. Finally, the fluorescence images were collected in parallelwith single point EEM spectra. From these studies, the effectiveness ofthe real-time implementation of fluorescence image collection,processing, and display with movement in the colon were assessed. Inaddition, sources of artifact present on the colonic mucosa such asmucous, stool, and prep were evaluated. Also, the anatomy of the colonmakes it desirable to collect images at large incident angles, and theeffectiveness of the moving average algorithm with these limitationswere determined. Finally, the intensities from fluorescence images werecompared to that from the single point optical fiber probes.

[0070] The excitation source used was a Coherent Innova 328. This laseris rated for 1 W in the UV, and requires 60 A at 208 V of electricalpower and 3 gal/min of water. The excitation light is coupled into anoptical fiber device including lengths of 12.5 and 16.5 m of fiber wererequired to deliver the excitation light to the distal end of thecolonoscope.

[0071] First, the excitation fiber must be incorporated in thecolonoscope. Next, a method is used to rapidly switch between whitelight and laser illumination. Finally, a method of quickly andaccurately registering the fluorescence results with the white lightimages must be implemented.

[0072] The colonoscopy procedures included prep of the patient with 3 ozof Fleet phospha soda mixed with 4 oz of water. There was no measurablefluorescence from the prep mixture using an optical fiber contact probeon the colonic mucosa in vitro with 370 nm excitation.

[0073] Using the electronic endoscope, white light reflectance andfluorescence images were collected sequentially in vivo during routinecolonoscopy. The white light image can include a vascular pattern ofarteries in red, and an outline of a vein in blue. Patches of specularreflection can be seen on the lower half of the images. The fluorescenceof normal mucosa appears uniform with an arterial pattern interspersedas reduced fluorescence intensity. This effect is attributed to theabsorption of fluorescence emission by hemoglobin. The vein does notappear on the fluorescence image, and there is virtually no specularreflection from the excitation light. The illumination filed onfluorescence is slightly smaller than that on white light, as depictedin FIG. 4.

[0074] An example illustrates the process of image collection,processing and evaluation of adenomatous polyps. A white lightendoscopic image taken of a sporadic polyp located in the rectum shows apolyp with visible architectural features about 5 mm in diameter islocated in the lower half of the image near the middle. In the rawfluorescence image the adenoma appears as a region of reduced intensitysurrounding a brighter central region.

[0075] This image was ratioed with its own moving average image, andmultiplied by 100 to produce the percent ratio image. Thresholds on theprocessed fluorescence images taken at 60%, 75%, and 90% were used todetermine the contour lines which define regions of mucosa with variouslikelihoods of containing dysplasia. The contours were then filled inpseudocolor to highlight areas of tissue to be targeted for biopsy. Thepseudocolors red, green and blue designate regions on the white lightimage which have high, medium and low probability, respectively. Thepolyp was found to be adenomatous on histology.

[0076] Overlay regions indicating disease included one located at thesite of the adenoma, and the other two corresponded to shadows cast bymucosal folds. The shadows appeared as regions of reduced intensity onthe fluorescence image. These effects were minimized by directing theendoscope normal to the mucosal surface. Moreover, the overlay regionswhich resulted from shadows changed in size and shape as the angle ofthe endoscope to the tissue surface varied, while those generated fromthe adenoma remained fixed in size.

[0077] White light and fluorescence images were collected from a totalof 30 patients undergoing routine colonoscopy, which included imagesfrom 14 adenomas and 5 hyperplastic polyps. A biopsy was taken of eachadenoma and one adjacent normal site. The fluorescence images wereprocessed by the moving average algorithm, and the sensitivity ofdetection was determined as a function of threshold values ranging from55% to 90%. The results of sensitivity are plotted in FIG. 8.

[0078] Autofluorescence images of colonic mucosa can be collectedendoscopically in vivo and can be used to identify and localizedysplasia in the form of adenomatous polyps. The SNR of the fluorescenceimages was typically above 30. The adenomas were correctly identified bythe fluorescence algorithm with high sensitivity. As shown in FIG. 8,the sensitivity of in vivo detection when the images are collected atnormal incidence is comparable to that from the in vitro studies. At athreshold of 75%, the sensitivity for detection of colonic adenomas was86%, compared to that of 92% for the in vitro experiments. In order todetermine the specificity, the true negatives and false positives mustbe identified. However, true negatives (false positives) correspond toregions of normal mucosa which were found to be normal (diseased) onfluorescence. These results were not obtained because additionalbiopsies incur additional risk of perforation. Furthermore, thefluorescence from hyperplastic polyps, which are not dysplastic, did notresult in regions of disease from the moving average algorithm.

[0079] In comparison of image size, the in vivo images encompassedregions of mucosa as large as 10×10 cm², whereas the specimens ofcolonic mucosa were only 2×2 cm² in the in vitro study. In such largefields of view, the colon contains many mucosal folds, and these layersof tissue blocked the excitation light from reaching theposteriorly-located normal mucosa, thus creating shadows. These foldswere not present in the in vitro studies. Diagnostic errors on theprocessed fluorescence image resulted primarily from these shadows. Thefluorescence method used is based upon the difference in intensitybetween normal and dysplastic mucosa. However, shadows appear as regionsof reduced intensity without dysplasia being present. This artifact canbe explained by the fluorescence excitation geometry. The fluorescenceexcitation is provided by one fiber located in the biopsy port forconvenience. The center of this instrument channel is 8.3 mm away fromthe center of the CCD detector. The white light image, on the otherhand, is illuminated by two fibers whose centers are located only 3.8 mmfrom the detector. Thus, the shadows on the white light image are muchless pronounced than those on fluorescence.

[0080] The fluorescence technique used a single fluorescence emissionband for detection of adenomas. This method worked well in vitro whenthe colonoscope is placed at normal incidence to the lesion, and nomucosal folds were present. However, during the clinical use of thefluorescence prototype, the view of the endoscope was often limited tothe side of the adenoma. Because the colon is a tube-shaped structure,some adenomas were anatomically located at sites where it was virtuallyimpossible to orient the colonoscopy at normal incidence to the lesion.As a result, one side of the lesion may not be surrounded by normalcolonic mucosa. Another situation was that the normal mucosa is far awayto produce fluorescence intensities sufficiently higher than that of theadenoma.

[0081] The fluorescence intensities were measured from the raw images.The normalized intensity values and the intensity ratios were taken atthree sites within the adenoma (denoted by left, center, and right inTable 3). The plot in FIG. 9 contains fluorescence intensity profilesthrough the adenoma, representing the raw fluorescence and percent ratiovalues, respectively. The adenoma was approximately 8 mm in diameter. Onfluorescence, the lesion is located between the 11 mm and the 19 mmmarkings on the abscissa, which are labeled by the vertical lines nearthe x-axis in FIG. 9. Most of the adenomas exhibited a singlefluorescence intensity minimum at the center of the lesion; the averageratio between normal and diseased pixels was 1.8±10.5 at the center, and2.0±0.6 and 2.0±0.7 at the left and right midpoints, respectively. Theaverage intensity ratio at these sites was 2.0±10.6. The results of thisprocedure show that the differences between normal colonic mucosa andadenomas for in vivo fluorescence images are very similar to that invitro.

[0082] Similarly, the fluorescence intensities were measured from theraw images for hyperplastic polyps. The normalized intensity values andthe intensity ratios were taken at three sites within the polyp (denotedby left, center, and right in Table 3). The plot in FIG. 10 shows thefluorescence intensity profiles through the hyperplastic polyp,representing the raw fluorescence and percent ratio values,respectively. The hyperplastic polyp was approximately 5 mm in diameter.On fluorescence, the lesion is located between the 17 mm and the 22 mmmarkings on the abscissa, which are labeled by the vertical lines nearthe x-axis in FIG. 10. The hyperplastic polyps exhibited anapproximately uniform fluorescence intensity across the lesion which wascontinuous with the normal colonic mucosa. The average ratio betweennormal and diseased pilxes was 1.1±0.1 at the center, and 1.2±0.1 and1.1±0.2 at the left and right midpoints, respectively. The averageintensity ratio at these sites was 1.1±0.2. Because this average ratiovalue is not significantly different from that of normal mucosa, it isnot surprising that no region of disease could be identified by thisintensity method.

[0083] In the in vivo images, the vascular pattern was clearly displayedon both the white light and fluorescence images. The vessels were notapparent on the in vitro images, perhaps because the blood supply of theliving colon was no longer intact. The hemoglobin in the blood is awell-known absorber of light, and produces linear patterns of weakfluorescence intensity. Thus, the intensities were measured from the rawfluorescence images of blood vessels. As shown in Table 3, the intensityratio from the blood vessels is 1.3±10.1. This value is significantlyless than the average from adenomas, thus blood vessels will not presentas a source of artifact on the overlay. Furthermore, image processingmethods can be used to remove the blood vessels based on their shape. InTable 3, the intensity ratios for adenomas, hyperplastic polyps, andblood vessels are summarized for comparison.

[0084] Endoscopic images and single point spectra can both providevaluable information about tissue biochemistry. Each method has its ownadvantages and disadvantages. The endoscope collects images, andprovides spatial information with sub-millimeter resolution. Thefluorescence intensity between normal mucosa and adenomas can becompared from the same image field within a fraction of a mm from eachother. Also, fluorescence images are collected remotely, thus thepressure on the tissue is uniform throughout the image field. However,it is more difficult to acquire spectral information with fluorescence.Because of the larger areas involved, the fluorescence energy may becometoo weak at each pixel to maintain sufficient SNR, unless very largeexcitation power is used.

[0085] On the other hand, single point optical fiber contact probescollect fluorescence from an area of approximately 1 mm in diameteronly. With an intensified optical multi-channel analyzer (OMA), spectraover a wide bandwidth can be measured with a good spectral resolutionand high SNR. However, the probe must be placed at several sites on themucosa to sample differences between normal and adenoma. Typically, thenormal mucosa sampled is several cm away from the adenoma, andcomparisons of the absolute intensity can be affected by biologicalvariability over distance.

[0086] The degree of contact of the probe on the polyp can vary duringthe in vivo measurements because the colonic musculature is constantlycontracting and expanding. As a result, movement is created which makesprobe placement difficult. The adenoma is round and slippery, and themovement of the colonic wall renders complete contact with the surfaceof the polyp very difficult. Furthermore, the distal end of the opticalfiber probe is not flat, but there is a 17° bevel. Thus, the orientationof the beveled side will affect the degree of contact as well.

[0087] Results of the colonoscopy procedure showed that it was verydifficult to place the probe onto the polyp for the 0.5 seconds requiredto collect a full EEM. Light escaping at various colors representing theexcitation sources was observed on the normal mucosa surrounding theadenoma. This observation suggests that the delivery of excitationenergy to the polyp and collection of fluorescence emission was notcomplete. Probe contact was hindered by the physiological movement ofthe mucosa, and by the fact that a flat probe was being placed on aslippery, hemispherical surface. Contact is not a problem for spectracollected on normal mucosa because this surface is flat.

[0088] Moreover, the ratios between the intensities of normal mucosa andadenomas can be affected by difference in the pressure exerted on eachsite. An in vitro experiment was conducted on a resected specimen ofcolonic mucosa which contained an adenoma. The fluorescence intensity inthe spectral range between 400 and 700 nm was measured as a function ofpressure exerted by the probe which was passed through the biopsychannel of a colonoscope. The pressure was measured with a balance. Asshown in FIG. 11, the fluorescence intensity increased with pressure,and the intensity ratio does not change if equal pressure is exerted onboth the normal and ademona sites. However, this is usually not the caseduring the clinical acquisition of spectra. The normal mucosa isrelatively flat, and measurements can be made with virtually completeprobe contact with a few grams of pressure. On the other hand, thepressure on the polyp cannot be made the same as that on the normal sitebecause the probe will slip off. The pressure on the normal site wasestimated to be about 5 grams, while that on the adenoma was estimatedto be close to zero. Thus, the difference in pressure exerted on thenormal mucosa and the adenoma may result in the intensity ratioincreasing from 2 to 3, as shown in FIG. 11.

[0089] Furthermore, on the recorded images of the colonoscopyprocedures, the normal mucosa showed an indentation at the site wherethe probe was placed during the collection of spectra. This observationconfirmed the estimate that several grams pressure was exerted on normalmucosa during data collection. On the other hand, the probe was seen toslide off the polyp when any significant pressure was exerted, whichresulted from the moistness of the surface. Thus, the pressure exertedon adenomas was significantly less.

[0090] Another procedure was conducted in vitro to compare thefluorescence intensity ratio between normal mucosa and adenoma asmeasured on imaging and single point. White light and fluorescenceimages of a resected specimen of colonic mucosa containing two adenomaswere obtained. The intensities were measured for 7 normal sitesimmediately adjacent to the adenomas on both imaging and single point.The results included the intensities that were normalized so that theaverage value is 100 for each system. This step allows for directcomparisons to be made at each point, and reveals that the intensitiesare within about 10% of each other. Furthermore, the normalizedintensity values range from 68 to 155 on imaging and from 63 to 136 onsingle point. Thus, the intensities-measured on normal mucosa depend onthe site sampled with both methods, and can vary by over a factor of 2.

[0091] In Table 4, the normalized intensities and the intensity ratiosare determined for the two adenomas on imaging and single point. Thesevalues are determined at the center and the left and right midpoints ofthe adenomas. For the left adenoma, the average intensity ratio was 1.43on imaging and 1.54 on single point. For the right adenoma, the averageintensity ratio was 1.52 on imaging and 1.72 on single point. Theseresults indicate there is little difference in the intensity ratiosbetween imaging and single point in vitro.

[0092] The fluorescence intensity ratio was calculated from Monte-Carlosimulations to determine the fluorescence intensity ratio, given thedifferent excitation and collection geometries of the imaging system andsingle point. In FIG. 12, a diagram of the collection geometry for theendoscope 100 and the single point probe 102 is shown. The endoscopecontains a 2.5 mm diameter objective lens 104, and is located in air ata distance 20 from the surface of the tissue. This geometry correspondsto a collection angle of 40°. The probe contains a quartz shield 106which is in contact with the tissue 16. The optical fibers are locatedat a distance of 2 mm from the tissue surface by this shield 106, andcollect light at a NA=0.22, which corresponds to a collection angle of12.7°. The optical parameters of colonic mucosa for the excitation andemission wavelengths are shown in Table 2.

[0093] The excitation used in the simulation is an infinitely-thin beamwith a divergence angle of 0°. The fluorescence intensity at a point onthe tissue from a uniform thick excitation beam can be determined fromthe fluorescence collected from a superposition of infinitely-thinexcitation beams which are incrementally displaced in distance from thepoint to be measured. However, this result is equivalent to integratingthe fluorescence intensity over the field of view. The LSF of the tissuefalls off quickly within several mm, thus the simulation integrates overa 2 mm region within the collection angle specified in Table 5. Theresults of the simulation are shown in terms of the intensity ratiobetween the light collected at the tissue surface with that of theexcitation. In Table 5, the intensity ratio between normal colonicmucosa and adenomas is 3.0 and 2.9 for the endoscope and the probe,respectively. The intensity ratio is similar for the endoscope and theprobe, a result which is consistent with the in vitro studies. Theintensity ratio for the endoscope is slightly higher than that of theprobe, which is consistent with the collection angle of the endoscopebeing smaller. Light from the highly fluorescent submucosa is morelikely to reach the detector with a smaller collection angle.

[0094] A model was developed to quantify the number of photons collectedby the endoscopic imaging system over the field of view at normal angleof incidence. This result is valid for both white light reflectance andfluorescence images, and can be applied to both the fiber optic imagingbundles and electronic imaging systems. The spatial distribution of theillumination and emission profiles of in the center and to fall offtowards the periphery of the image. When combined with the detectornoise statistics, the SNR of the image can also be determined. Thisanalysis showed that distance and optical collection geometry produces aprofile in which the SNR at the periphery was always lower than that inthe center. This parameter is needed for developing algorithms foridentifying tissue lesions. Also, the collected light intensity wasfound to decrease with the square of the distance between the distal endof the endoscope and the tissue. Furthermore, the light collection bycoherent imaging bundles is limited by the numerical aperture of theoptical fiber. This analytic tool can be used to design the opticalparameters of the fluorescence imaging system and to identify the typeof light source required to excite the fluorescence.

[0095] The methods developed for endoscopic imaging model were used todetermine the excitation source, optics, and detectors necessary forbuilding two fluorescence imaging systems. The first design consisted ofa fiber optic colonoscope which detected the fluorescence image at theproximal end with an intensified CID camera. A 400 nm long pass filterwas used to block the reflected excitation light, and a quartz opticalfiber located external to the colonoscope was used for image excitation.The second design was a modification of the first to accommodate therequirements for clinical use. This system used an electroniccolonoscope with dual instrument channels, and detected fluorescenceimages at the distal end. The cutoff in spectral sensitivity of the CCDdetector below 400 nm was used to avoid the reflected excitation light.An illumination probe with a high NA quartz optical fiber was coupled toa microlens and inserted into one instrument channel for imageexcitation. In both systems, the excitation source was an argon-ionlaser which delivered about 300 mW at λ_(ex)=351 and 364 nm, andmicrocomputer with a frame grabber was used to acquire, process, anddisplay the diagnostic images.

[0096] Autofluorescence images from human colonic adenomas werecollected with the fiber optic system with high SNR in vitro. For widearea surveillance of the colon wall, regions of mucosa as large as 100mm² must be illuminated. Furthermore, the endoscopic images arecollected remotely, and the intensity collected falls with distance dsquared. Previously, fluorescence spectra were collected from contactprobes which illuminated an area of about 1 mm². The results of thisstudy showed that excitation sources, optics, and detectors used in thisdesign could collect autofluorescence images with sufficient SNR todistinguish between normal colonic mucosa and adenomas. In the fiberoptic system, an SNR of over 30 was attained, which exceeded the minimumSNR requirement of 7.

[0097] Fluorescence images were then collected from samples of resectedcolonic mucosa in vitro to evaluate the potential use of this techniquefor wide area surveillance of dysplasia Colectomy specimens from threepatients with familial adenomatous polyposis containing polypoid andnon-polypoid adenomas were studied. Each raw image was corrected fordifferences in distance and instrument light collection efficiency bynormalizing to a spatially averaged image. Intensity thresholding wasthen used to identify diseased regions in mucosa. The sensitivity andspecificity for detecting a region of dysplasia depended on thethreshold value selected. With the threshold set to 75% of the averagenormal intensity, a sensitivity of 90% and a specificity of 92% wereachieved. The average fluorescence intensity from normal mucosa wasfound to be greater than that from the adenomas by a factor of 2.2±0.6.These results demonstrate the potential of this technique to directbiopsy site selection.

[0098] The results from the in vitro studies provided motivation forconducting an in vivo study. The electronic system was used to collectautofluorescence images from colonic adenomas in vivo. In the thissystem, an SNR of over 30 was attained as well, which exceeded theminimum SNR requirement of 4. Fluorescence images were collected from 14adenomas and 6 hyperplastic polyps from 30 patients undergoing routinecolonoscopy. The fluorescence images were collected in a 33 ms frames,and were processed by dividing the raw fluorescence image with a movingaverage image. The processed images displayed regions of mucosa with aprobability of containing dysplasia in the form of adenomas, as verifiedon histology. With the threshold set to 75% of the average normalintensity, a sensitivity of 86% was achieved for detecting adenomas anda specificity of 100% was attained for hyperplastics. On average, theratio between the fluorescence intensity of normal mucosa to that fromadenomas was 2.0±0.6 and to that from hyperplastic polyps was 1.1±0.2.The diseased regions on fluorescence best corresponded to the adenoma onwhite light when the colonoscope was at normal incidence. At higherangles there were greater effects from shadows. These results showedthat dysplasia can be identified on fluorescence images in vivo.

[0099] In the single point optical fiber contact probe studies theaverage intensity ratio between the fluorescence at 460 nm from normalcolonic mucosa and adenomas was found to be about 3, while that inendoscopic imaging this ratio was measured to be 2.0+0.6. Directcomparison of fluorescence imaging and single point measurements invitro revealed that there was little difference between the intensityratio measured on imaging compared to that measured from single point.There are two possibilities that can account for the difference inintensity ratio between the two methods. First, the ratio of 3 measuredby the single point method was performed in vivo. A lower ratio may haveresulted in vitro because of the loss of blood flow, which is known toabsorb light.

[0100] Alternatively, the difference in the ratios may result fromcontact and pressure artifacts. Videotapes of the colonoscopy procedureshowed that it was very difficult to place the probe onto the polyp forthe 0.5 seconds required to collect a full EEM. Light at various colorsrepresenting the excitation sources was observed, which indicated thatthe delivery of excitation energy to the polyp and collection offluorescence emission was not complete. Probe contact was hindered bythe physiological movement of the mucosa, and by the fact that a flatprobe was being placed on a slippery, hemispherical surface. Contact isnot a problem for spectra collected on normal mucosa because thissurface is flat. Furthermore, increased pressure was found to elevatethe fluorescence intensity collected. Higher pressures were exerted onthe normal mucosa compared to that on the polyp. The probe was seen toslide off the polyp when any significant pressure was exerted. Bothdifferences in contact and pressure in vivo resulted in a higher ratiobetween normal mucosa and adenoma. On the other hand, the fluorescenceimages are collected remotely, and the pressure and contact parametersare identical for normal mucosa and adenoma.

[0101] Finally, the results of the clinical studies identified futuredirections to improve the sensitivity and clinical usefulness offluorescence endoscopic imaging. The shadow artifact can be reduced byilluminating the tissue through the two white light ports. Thismodification can be accomplished by replacing the glass fibers withquartz, thus allowing for both white and excitation light to betransmitted. Furthermore, the shadow artifact, angle of incidence, anddetection yield can all be improved by collecting multi-spectral imagesconsisting of two or more fluorescence images. Lastly, the concurrentcollection of EEM spectra can be used to identify new excitationwavelengths which result in higher intensity contrast ratios.

[0102] Dysplastic tissue exhibits an increase in red fluorescence whichcan be detected to improve the sensitivity of disease detection. Thusanother embodiment includes the collection of multiple emissionwavelengths. One method of collecting multiple fluorescence emissionwavelengths is to use an electronic endoscope (e.g. Olympus, Model CF IOOTL) with a CCD detector which is sensitive to the red, green, and blue(RGB) regions of the visible spectrum. Fluorescence images from each RGBframe can be captured and processed, providing more detailed informationfor use in a diagnostic procedure. Furthermore, the use of spectrallineshape information from images at different wavelengths reduces allgeometric distortions. The TI TC244 has a quantum efficiency of 30% at640 nm and 15% at 480 nm [TI Mannual, 1994]. Extrapolating from the 370nm imaging data and the EEM data, the SNR of 10:1 in the red and 50:1 inthe blue is anticipated.

[0103] Performing the detection on the distal end of the electroniccolonscope has many practical advantages. First, the same detector canbe used for both white light and fluorescence imaging. A single detectornot only results in a perfect registration of the two images, but avoidsthe need to interchange of cameras, which can be cumbersome. Second,fewer optical elements results in a transmission efficiency offluorescence photons which is significantly higher than that of a fiberoptic imaging bundle. Third, the packing geometry of CCD pixels allowfor minimal loss of surface area of detection, unlike fiber opticimaging bundles which have a hexagonal packing array.

[0104] While there are advantages to detecting the fluorescence imagewith a distally located CCD, a fiber optic imaging bundle with proximaldetection has advantages as well. The spectral bands of the distal CCDis limited to the RGB response of the distal detector, while thefluorescence collected by a fiber optic imaging bundle could be filteredinto an unlimited number of spectral images. Also, detection of thefluorescence image at the proximal can allow for detection with a gatedintensifier. This device enables use of pulsed lasers.

[0105] The EEM study provides valuable guidance about new imagingstrategies. The results indicate that excitation near 410 nm is useful.The contrast between normal and adenoma tissues provided by the bluefluorescence is greatly enhanced compared to that obtained with ourcurrent excitation wavelength (10:1 versus 2:1). In addition, the redfluorescence is quite pronounced for adenoma. Extrapolation of theconclusions of the morphological model developed using λ_(ex)=365 nm tothis new excitation wavelength suggests that the blue fluorescencecontains information about both crowding of the crypts and mucosalthickness, and that the red fluorescence contains information aboutcrypt cell dysplasia. Hence, collecting images at red and blue emissionwavelengths should provide both high contrast diagnostic images andsignificant new histological information. In addition, the ratio imagecan be used to normalize out shadow effects. The next phase of theimaging studies will use 410 nm excitation. A krypton ion laser(Coherent Innova Model 302) will provide 500 mW of power at the twolines 407 and 413 nm. This level of power is adequate to achieve largefluorescence signals in both red and blue bands. This laser will beinstalled at the BWH Laser Laboratory along with the existing 365 nmargon ion laser.

[0106] In addition, multiple excitation wavelengths can be employed. Oneapproach would be to use excitation from the 407 and 413 nm lines of akrypton ion laser to excite the red fluorescence and to retain the 365and 351 run lines from argon ion laser to excite the blue fluorescence.Two hardware configurations include (1) a fiber endoscope with aswitchable filter wheel between the scope and camera, and (2) adual-chip endoscope. Such a system has been developed, for example, byAmerican Hospital, Inc., for stereo viewing during endoscopy. One canmodify one of the windows on the chip with a spectral cut-off mechanism.The timing of the red-sensitive imaging channel can be synchronized withthe excitation light.

[0107] The diffuse reflectance image at 407-413 nm can be explored toobtain information about the tissue hemoglobin content. This image canbe obtained by installing a filter with the appropriate bandwidth on therotating wheel in front of the white light source. The approach is toratio this reflectance image with the fluorescence images in the red andblue frames. In order to develop the required algorithms, and to decidehow to optimize the spectral information collected, an extensive contactprobe study with 410 nm excitation can be performed.

[0108] The shadow artifact obtained using the broadband intensityalgorithm with 365 nm excitation can be greatly reduced by use of animproved excitation geometry. Currently, excitation light is deliveredthrough a single quartz fiber located in the biopsy channel located 8.3mm from the CCD detector. The use of a single illumination beam locateda large distance from the CCD chip tends to enhance shadows. Incontrast, in the conventional while light images produced by thiscolonscope, shadows are minimized by use of two closely spaced whitelight illumination beams symmetrically positioned on opposite sides ofthe CCD chip. By replacing the illumination fibers with quartz fibers,the UV light can be delivered through the two white light illuminationports, which are located only 3.8 mm from the CCD detector. Implementingthis requires modifying the video processor to enable alternate couplingof white light and laser excitation into the illumination fibers.

[0109] Other spectral endoscope improvements can include: (i) regulatingthe excitation light intensity on the tissue surface via feedbackcontrol. This provides constant illumination, regardless of viewingdistance, and is also important for patient safety; (ii) minimizing thestreaking effect of the fluorescence excitation on the white lightendoscopic imaging display by timing the fluorescence excitation tooccur during the “blank” periods of the filter wheel used in theendoscope white-light source. Feedback control of the excitation lightcan be accomplished by measurement of the average intensity on thefluorescence image. The intensity of this average value will be used tomodulate the open period on the shutter or filter wheel. The streakingeffect can be completely removed by implementing the identical filterwheel for blocking the excitation light that is used for producing theRGB illumination on the white light mode.

[0110] As described above, a large argon-ion laser was used as anear-LTV excitation source for imaging studies. Although adequate forthese studies, this light source is expensive and bulky and operates atonly a few discrete wavelengths. Such a laser system with its specialelectrical and water cooling requirements cannot be easily moved,preventing use at multiple sites. Alternative excitation sources can beconsidered which include a pulsed laser and a white-light source withfilters, both of which are compact and transportable.

[0111] For applications in which near-UV excitation is appropriate, apulsed ND:YAG laser is used because it can provide third harmonicradiation at 355 mm with sufficient average power for spectral imaging.In both the in vitro and in vivo studies, good SNR was obtained with 300mW of laser power, which corresponds to 10 mJ of energy per frame.Therefore, a frequency tripled ND:YAG laser with a 5-10 ns pulseduration operating at 30 Hz with an average power of 300 mW at 355 nmwill be adequate. Using a CCD camera gated at about 10 ns, this shortexcitation pulse enables simultaneous acquisition of white light andfluorescence images. Within this short temporal gate the white lightbackground is negligible, obviating the need to chop the white lightillumination. There are no special power or water requirements forlasers of this type and a fluorescence endoscope system with such alaser will be easily transportable.

[0112] A mercury lamp can also be used as an excitation source. Such asource is compact and lightweight and can provide a bright, narrowbandillumination at a number of excitation wavelengths. Employing this lightsource simplifies system design and reduce cost, enabling less expensiveunits to be produced for use at multiple sites. The key issue is whetherenough light in the desired wavelength range can be coupled into theillumination fiber(s). A commercial white light source with a 150 Wxenon lamp is capable of delivering as much as 80 mW of white light atthe distal end. Utilizing a 50 run excitation bandwidth, about 20 mW oflight can be used to induce tissue fluorescence.

[0113] At selected wavelengths, mercury lamps have 5 to 10 times higheroutput powers than that of xenon. This indicates that with a 500 Wmercury lamp having a relatively small filament, at least 300 mW ofuseful excitation light should be available at the distal end of theillumination fibery should be sufficient for collection of good qualityfluorescence images from colonic tissue. In addition, to further enhanceSNR, either the total area of illumination can be reduced or imagingelements can be binned together. A lamp and power supply can be selectedfor this application with the proper brightness, stability and minimumelectrical interference.

[0114] Currently, the image processing scheme is based on ratioing theraw image to a spatially-averaged image, and applying a thresholdcriterion for classifying a region of tissue as normal or diseased. Theaveraging window and detection threshold values are pre-flexed,regardless of the polyp size, viewing angle and distance. Thepredetermined values limit the range of polyp sizes which can beaccurately measured. Improved image processing and thresholding methodswill employ variable window sizes for spatial averaging and variablethresholds. Information from the raw digitized image about the diameterof the largest lesion in the image will be used to determine theseparameters. This change in the window size as a function of the lesionin the image field will maximize the intensity ratio and optimize theperformance of the fluorescence method.

[0115] Image analysis methods for extracting information frommultivariate images can also be explored. A multivariate image is acollection of congruent images of the same object measured withdifferent variables, such as reflected wavelengths, or fluorescence orRainan band intensities. Many methods are available for analyzingmultivariate images, and they can be adapted to image analysis. Ingeneral, three steps will be followed, image processing, objectsegmentation, and contrast measurement. The images will first beprocessed based on the selected operation, such as moving-windowaverage, intensity difference or ratio. The processed image will then besegmented based on both frequency and intensity information. This can bedone either through thresholding, quick/slow descent, or region growth.These methods can be coupled to the concomitant identification anddisplay of a lesion(s) based on a probabilistic scheme.

[0116] When techniques for collecting multiple spectral images aredeveloped and a database of such images are built, more advanced imageanalysis methods, such as principal component analysis and regressionanalysis can be used. Principal component analysis does not assume aknown (a priori) distribution, but instead employs a set of calibrateddata to extract information about structures exhibiting pre-malignantchanges. The regression technique is based on the principle of buildingup a mathematical relationship between two groups of variables, i.e.,between a number of independent variables and one or more dependentvariables. As an example, a logistic regression to correlate spectralintensities in the images with histopathology of dysplastic lesions.

[0117] The development of the fluorescence imaging endoscope hasdemonstrated the potential to perform wide-area surveillance colonscopyusing fluorescence. The fluorescence image can be analyzed in real timeand can provide the endoscopist with an instant interpretation of theprobability of dysplasia determined using a previously-validatedalgorithm. In addition, the ability to guide biopsy can be used with thepresent invention. In patients with FAP, fluorescence imaging can beused to direct mucosal biopsies to areas that are endoscopicallynormal-appearing (non-polypoid) but, based on their spectralcharacteristics, can have an increased likelihood of being dysplastic.Histopathological assessment of mucosal biopsies will be correlated withspectral data to validate for detection of “flat” dysplasia.

[0118] The following method can be followed for determining thecapability of the fluorescence imaging system for directing biopsy. Theentire surface of the colon wall, both at colonscopy and using resectedsamples at colectomy, is systematically imaged, and isolated areas whichare diagnosed as dysplastic selected for directed biopsy. Random areasdiagnosed as benign can also be sampled and the spectral diagnosisconfirmed by histological analysis. Again, the effects of complicationssuch as inflammation can be investigated. Once an imaging algorithm hasbeen validated, it can be adapted to the detection of dysplasia inpatients with UC. As in the case of the contact probe studies,diagnostic algorithms for UC must be capable of evaluating patients withvarious degrees of background inflammation. The same patient groupsstudied with contact probe EEMs will be studied with fluorescenceimaging. An important potential benefit of wide area fluorescencesurveillance is that one or more of the otherwise random biopsiesobtained during conventional surveillance colonscopy may be directed bythe results of fluorescence imaging. Those biopsies can be separatedfrom the remainder of the random biopsies to assess whether fluorescenceimaging can increase the yield of dysplasia detection over randomsampling.

[0119] The development of the rapid EEM and spectral imaging systemsrepresent two very important advances in instrumentation. Two systemsare complementary. The imaging system views wide areas of mucosa in realtime, and the EEM system provides complete spectral characterization ofa given site of colonic mucosa. The two instruments can be usedsimultaneously, where appropriate. The EEM probe is placed through thesecond channel of a two channel colonscope. Thus, each system can beused to verify the other. Also incorporated herein is the publicationattached hereto and entitled “Real-Time in vivo endoscope imaging offluorescence from human colonid adenomas.”

[0120] The following table compares the signal size expected for whitelight imaging, fluorescence imaging observed with the endoscope CCD, andthe fluorescence imaging using the optics module with the intensifiedCID camera. The parameters listed below are taken from either themanufacturer's specifications or from experimental measurements. TABLE 1Pentax Imaging Device Definition Pentax (white light) (fluorescence) UVModule λ_(ex) (nm) ex wavelength 356 356 λ_(em) (nm) ex wavelength 460460 Δλ (nm) em bandwidth 400-700 400-700 400-700 P_(o) (mW) power 1 300300 Δt (s) integration time 0.011 0.011 0.033 d (mm) distance 20 20 20Diameter (mm) area illum 70 70 28 θ_(m) (degrees) max angle 60 60 35N_(f) number 88560 88560 10000 (pixels/fibers) r_(L) lens (mm) radius1.25 1.25 0.3 ε_(t) tissue 1 5.00E−05 5.00E−05 efficiency f_(o) packing1 1 0.6 fraction T_(f) % trans filter 1 1 0.8 T_(i) % trans 1 1 0.9imaging T_(o) % trans optics 1 1 0.9 η_(s) photocathode 0.2 0.2 0.1 eff.g group factor 1 1 1 N_(s) signal photons 1.7 × 105 2500 338 σ_(e)electronic noise 55 55 50 G gain 1 1 10,000 SNR signal/noise 407 34 18

[0121] TABLE 2 normal adenoma λ (nm) λ_(ex) = 370 λ_(em) = 460 λ_(ex) =370 λ_(em) = 460 μ_(a) 0.9 0.45 2.1 1.1 μ_(s) 15.0 9.5 8.5 5.9 g 0.9 0.90.9 0.9 mucosal thickness (μm) 450 450 1000 1000 quantum eff (mucosa)0.1 0.1 0.8 0.8 quantum eff (submucosa) 0.1 0.1 0.8 0.8

[0122] TABLE 3 Adenoma Hyperplastic Blood Vessels Normalized IntensityNormal 100.0 ± 27.5  100.0 ± 18.1  100.0 ± 16.1  Left 54.0 ± 16.3 87.9 ±16.3 Center 59.9 ± 16.7 95.9 ± 16.7 75.3 ± 5.1  Right 54.4 ± 17.7 98.1 ±17.7 Intensity Ratio (normal/lesion) Average 2.0 ± 0.6 1.1 ± 0.2 1.3 ±0.1 Left 2.0 ± 0.6 1.2 ± 0.1 Center 1.8 ± 0.5 1.1 ± 0.1 Right 2.0 ± 0.71.1 ± 0.2

[0123] TABLE 4 Imaging Single Point Normalized Intensity NormalizedIntensity Intensity Ratio Intensity Ratio Left Adenoma Left 65 1.54 581.72 Center 74 1.35 74 1.35 Right 71 1.41 65 1.54 Avg. 70 1.43 66 1.54Right Adenoma Left 61 1.64 57 1.75 Center 81 1.23 67 1.49 Right 59 1.6952 1.92 Avg. 67 1.52 59 1.72

[0124] TABLE 5 endoscope probe normal adenoma normal adenoma d (mm) 2020 2 2 θ_(m) (deg) 4 4 12.7 12.7 n₀ 1.0 1.0 1.4 1.4 n_(t) 1.4 1.4 1.41.4 370 2.8E−02 2.8E−02 4.2E−03 6.2E−04 460 8.8E−05 2.9E−05 2.8E−039.7E−04 Ratio 3.0 2.9

[0125]FIG. 13 presents a schematic outline of the system which has beendemonstrated in clinical practice in a format which will allowcomparison with the improved systems to be described below. Theembodiment shown uses an ultraviolet laser source 200, switched by ashutter 202 and focused with a lens 204 into a fused silica fiber probe206 inserted into a biopsy channel of an endoscope 208 to deliver it toa tissue site 210 so that it can illuminate the tissue over an area 212.The UV illumination thus comes from an aperture 214 which is differentfrom the endoscope's own illumination ports 216. In the dual-channelPentax endoscopes used in the clinic this procedure leaves one biopsychannel 218 free.

[0126] The endoscope camera 220 obtains its white light illuminationthrough its own fiberoptic illuminator 222 from a broadband Xenon arclamp 224 and collection optics 226. A non-standard shutter 228 undercomputer 230 control 232 is attached to allow the white lightillumination to be turned off while fluorescence images are being taken.The fluorescence image signal 234 is processed by the endoscope's videoprocessor 236 to produce a standard video signal 238 which is digitizedby a framegrabber in computer 230. The processed image signal 240 withits information on the state of the observed tissue is sent to monitor242. The entire diagnostic procedure is initiated by a foot switch 244attached to the computer by a cable 246.

[0127]FIG. 14A shows a design for the fluorescence imaging system whicheliminates the tendency of the previous system to identify shadows inthe image as regions of dysplasia. The improved design uses a 100Wmercury (Hg) arc lamp light source 302, dichroic mirrors 304 and 306,wavelength filters 308 and 310 and rotating shutters 312 and 314 toprovide precisely-timed, tissue-illumination pulses in two separatewavelength bands. The first wavelength band is centered on thenear-ultraviolet (365 nm) mercury resonance line and is used to obtainthe UV autofluorescence image. The second wavelength band is at the endof the visible spectrum and is used to obtain a simultaneous ornear-simultaneous, reflectance image for the purpose of identifyingshadows and the extent of the UV illumination field.

[0128] A reflectance (non-fluorescing) image taken with an endoscopecamera system measures the brightness of the tissue surface 316 in itsfield of view. To the extent of the tissue surface is a Lambertian(non-specular) reflector (generally the case) this image indicates thedistance of the tissue from a single illumination source (or a weighteddistance from multiple sources). If these illumination sources are notin the direct line-of-sight from the camera to the tissue source therewill be shadows. A reflectance image can thus be used to measure boththe UV illumination 318 at the tissue surface and the presence ofshadows in the fluorescence image as long as the UV illumination and thevisible illumination emanate from the same aperture 320 with the sameangular divergence. Note that this condition can be satisfied either bya two-color illumination fiber 322 passed through a biopsy channel of anendoscope 324 or by the two-color illumination being passed through theillumination bundle 326 of the endoscope. A shutter 328 switches off thenormal white-light illumination of the endoscope while the twodiagnostic images are being obtained. The closing of shutter 328 undercomputer control 329 occurs at the same time as the opening of shutter330 by control line 331. This action enables the two-color light toreach the fiber 322 and thus the tissue 316.

[0129] The algorithm for using the visible reference image along withthe fluorescence image is as follows. The video signals 332 from the CCDcamera at the distal tip of the endoscope 324 are converted by the videoprocessor 334 to a standard NTSC color video signal 336 and sent to avideo framegrabber in computer 338. The two images are first correctedfor the gamma factor applied to the video signal by the video processorto insure that the digitized images acquired by the framegrabber in thecomputer are linear measurements of the tissue surface brightness. Thisis accomplished in real time by the framegrabber input look-up table.The two images are then normalized to their peaks, which will generallybe a region of non-dysplastic tissue in the visual field. Thisnormalizes the two illumination fields. On a pixel-by-pixel basis thefluorescence image value is then divided by the visible reference imagevalue. If the ratio falls below a predetermined threshold (typicallyon-half to one-third) then that pixel in the image represents a regionof reduced fluorescence which is indicative of dysplasia. This pixel canthen be set to a false color state in an output video signal 340 sent toa monitor 342 to indicate to the clinician the probability of dysplasia.A prior threshold requirement on both images insures that the ratioobtained is significant and eliminates false color output in regions ofshadow of low illumination at the edges of the video field. This entireoperation occurs for every depression of the footswitch 344 which isconnected to the computer through cable 346.

[0130] In the improved design both the UV-excitation light pulse and thevisible-reference light pulse are delivered to the tissue through thesame optical fiber 322 inserted through a biopsy channel of theendoscope. The condition that the two illumination sources have the sameangular distribution is assured by the design of the light collectionapparatus shown in FIG. 14A. A single Hg arc lamp 302 is used as thesource of both wavelengths. A dichroic mirror 304 reflects the UVportion of the spectrum and transmits the visible portion. Filters ineach path further refine the bandwidth of the two beams. The UV filter308 must reject visible light to a high degree since the efficiency ofthe 460 nm tissue fluorescence is only about 0.1%. The filter 310 in thered path is less critical but the chosen center wavelength should avoidhemoglobin absorption bands to provide the best reference image. Notethat the design of the beam splitting optics and beam combining opticshave an even number of reflections in both the UV and visible arms. Thisassures that any angular deviations of the output beams due to motion ofthe lamp track each other. It also makes the directions of the outputbeams invarient under translations and small rotations of the beamsplitting and recombining optics as a whole.

[0131] In the improved source design the current through the Hg lamp 302is boosted at appropriate moments to increase the lamp output power forthe UV exposure. This allows a larger area of tissue to be scanned fordysplasia in a single image. The data in FIG. 14B show that the UVoutput power from a 100 W Hg lamp is a linear function of its inputpower to at least a factor of 3 over its nominal rated power. Since thelamp discharge maintains a constant voltage drop across the arcregardless of current, the lamp output power is essentially proportionalto current. At least 50% power to the lamp must always be maintained,however, to keep the mercury in the vapor phase. The lamp power supply348 in the improved fluorescence system utilizes a DC current section tomaintain the idle current and a computer-controlled 350, pulsed currentsection which can rapidly switch in multiple constant-current sources tovary the output power of the lamp as required by the imaging system. Ifthe idling power is kept below the rated power and the current pulsesare kept to a sufficiently small duty factor, then the pulsed UV outputcan be sustained continuously.

[0132] The rotating shutter in the UV path 312 and in the red path 314of the light source are designed to provide pulsed illumination lightaccording to the timing diagram in FIG. 14C. This diagram shows how thisfluorescence imaging system is used with a monochrome camera, which usesa xenon arc lamp 352 for illumination and a rotating blue-green-redfilter wheel 354 to synthesize a color image from 3 monochrome images.In this type of system a pulse of blue light first illuminates thetissue for about 6 milliseconds and the resulting tissue reflectanceimage is digitized for the next 6 milliseconds. The illumination must beturned off during the readout period because the monochrome camera usedcontinues to collect photo current in the pixels as they are lineshifted to the readout electronics. Illumination during the readoutcauses a smearing artifact in the image. The UV illumination is switchedin during the normal blue exposure period and the red light illuminationis switched in during the red exposure period. The green exposure periodis not generally used but could be used to obtain an additionalreference image or an additional UV fluorescence image. The shutters aretimed to the video acquisition system using an LM1881 Video SyncSeparator circuit to develop and even/odd frame synchronization pulse356 from the standard composite video output signal. Phase-locked-loops(PLL) and 358 and 360 synchronize the phase of the chopper wheels tothis signal by varying a voltage to their DC driving motors 362 and 364.This signal is also used to synchronize the current pulser 348. In theschematic of FIG. 14A the chopper wheels are shown in collimatedportions of the beam. In practice, these chopper wheels are placed at aninternal focal point in the two arms of the optics train (notillustrated) to provide for fast rise and fall times for the lightpulse.

[0133] Note that the dual-wavelength illumination method can also beused with standard, color-CCD camera endoscopes. In this case, the UVlight illumination and red light reference illumination are presentsimultaneously. The UV-induced fluorescence (primarily at 460 nm) isthen detected by the blue-responsive pixels in the CCD camera and thereference reflectance image is detected by the red-responsive pixels.Note that the visible blue light must still be removed from thediagnostic illumination so as not to decrease the contrast of thefluorescence image. The slight amount of red tissue fluorescence seen indysplastic tissue due to the UV excitation is much smaller than thelevel of direct red illumination. The slight increase also acts toreduce the fluorescence/reference ratio which properly increases(slightly) the measured probability of dysplasia.

[0134]FIG. 15 shows a preferred embodiment of the fluorescence imagingsystem in which dual wavelength illumination capability as well aswhite-light illumination capability is built into the video endoscopesystem itself 400. This requires the illumination bundle 402 of theendoscope 404 to be transmissive at UV wavelengths which is not usuallythe case with current commercial systems. Such a design fulfills thedesign requirement that the UV-excitation and visible-referenceillumination emanate from the same aperture or apertures 406. Such asystem would also be easier for an operator to use since theillumination fiber would not have to be threaded down through a biopsychannel 408 and those channels would be free for their standard uses.The tissue surface 410 would be illuminated over a larger area 412 withfewer shadows since dual illumination ports 406 are standard. The videosignal 414 would be processed by the endoscope system 416, formattedinto a standard signal 416 and processed by the computer 420. The outputvideo signal with the false color overlay 422 would be sent to a monitor424 for the clinician to see in real time. The diagnostic illuminationwould be initiated by a footswitch 426 connected by a cable 428 to thecomputer 420 with the light pulses controlled by signal lines 430 and432 to the shutters in the light source.

[0135] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

What is claimed:
 1. A method of fluorescence imaging comprising:providing an endoscope having an optical guide that is optically coupledto a first light source and a second laser light source, the endoscopehaving an image sensor at a distal end; collecting a reflectance imagewith the image sensor and generating a reference; collecting afluorescence image with the image sensor; and processing thefluorescence image with the reference to provide a processedfluorescence image.
 2. The method of claim 1, further comprisingproviding an image sensor having a filter to remove ultraviolet lightfrom light returning from tissue.
 3. The method of claim 1, wherein theimage sensor has a sensitivity in the ultraviolet region that is lessthan half of the sensitivity of the sensor in a visible region.
 4. Themethod of claim 1, wherein the reference corrects intensity for shadowsin the fluorescence image.
 5. The method of claim 1, wherein thefluorescence image is non-intensified.
 6. The method of claim 1, whereinthe first light source is a broadband source.
 7. The method of claim 1,wherein the second light source generates a wavelength in the range of350 nm to 420 nm.
 8. The method of claim 1, further comprisingcompensating for shadows on a tissue surface to be imaged.
 9. The methodof claim 1, wherein the first and second light source are opticallycoupled to the same optical guide.
 10. The method of claim 1, furthercomprising providing a shutter coupling each light source to an opticalfiber.
 11. The method of claim 1, further comprising imaging dysplasiaon a tissue surface.
 12. The method of claim 1, wherein the image sensorcomprises a color-sensitive image sensor.
 13. The method of claim 12,wherein the image sensor comprises a color charge coupled device (CCD).14. The method of claim 1, wherein the image sensor detects red, greenand blue wavelengths.
 15. The method of claim 1, wherein the imagesensor comprises a pixellated flat panel detector.
 16. The method ofclaim 1, wherein the step of collecting a fluorescence image comprisescollecting at least three wavelengths in a range of 400-700 nm.
 17. Afluorescence imaging system comprising: an endoscope; a light sourcecoupling to an optical guide extending through the endoscope; and animaging sensor at a distal end of the endoscope that detects afluorescence image and reflectance image of tissue.
 18. The fluorescenceimaging system of claim 17, wherein the light source comprises a firstbroadband light source and a second narrow band light source.
 19. Thefluorescence imaging system of claim 18, wherein the narrow band lightsource emits light having a wavelength in the range of 350 nm to 420 nm.20. The fluorescence imaging system of claim 17, further comprising afiber optic device optically coupled to the light source.
 21. Thefluorescence imaging system of claim 17, further comprising a processorthat processes the reflectance image and the fluorescence image andgenerates compensated fluorescence image.
 22. The fluorescence imagingsystem of claim 17, further comprising a shutter positioned along anoptical path between the light source and an optical fiber extendingthrough the endoscope.
 23. The fluorescence imaging system of claim 17,wherein the image sensor has reduced sensitivity in an ultravioletspectral region relative to sensitivity in a visible spectral region.24. The fluorescence imaging system of claim 17, wherein the imagesensor comprises a filter reducing image sensor sensitivity below 400nm.
 25. The fluorescence imaging system of claim 23, wherein the sensorsensitivity in the ultraviolet spectral region is less than one half ofthe sensitivity in the visible region.
 26. The fluorescence imagingsystem of claim 17, wherein the imaging sensor comprises acolor-sensitive image sensor
 27. The fluorescence imaging system ofclaim 26, wherein the imaging sensor comprises a color charge coupleddevice (CCD).
 28. The fluorescence imaging system of claim 17, whereinthe imaging sensor detects red, green and blue wavelengths.
 29. Thefluorescence imaging system of claim 17, wherein the light sourcecomprises a laser light source.
 30. The fluorescence imaging system ofclaim 17, further comprising a processor that processes saidfluorescence image and said reflectance image to produce a processedoutput image.
 31. The fluorescence imaging system of claim 17, furthercomprising collecting three wavelengths in a range of 400-700 nm. 32.The fluorescence imaging system of claim 17, wherein the imaging sensorcomprises a pixellated flat panel detector.